Stability and Cell Permeability of Sulfonyl Fluorides in the Design of Lys‐Covalent Antagonists of Protein‐Protein Interactions

Abstract

Recently we reported on aryl-fluorosulfates as possible stable and effective electrophiles for the design of lysine covalent, cell permeable antagonists of protein-protein interactions (PPIs). Here we revisit the use of aryl-sulfonyl fluorides as Lys-targeting moieties, incorporating these electrophiles in XIAP (X-linked inhibitor of apoptosis protein) targeting agents. We evaluated stability in buffer and reactivity with Lys311 of XIAP of various aryl-sulfonyl fluorides using biochemical and biophysical approaches, including displacement assays, mass spectrometry, SDS gel electrophoresis, and denaturation thermal shift measurements. To assess whether these modified electrophilic "warheads" can also react with Tyr, we repeated these evaluations with a Lys311Tyr XIAP mutant. Using a direct cellular assay, we could demonstrate that selected agents are cell permeable and interact covalently with their intended target in cell. These results suggest that certain substituted aryl-sulfonyl fluorides can be useful Lys-or Tyr-targeting electrophiles for the design of covalent pharmacological tools or even future therapeutics targeting protein-protein interactions.

Design, Synthesis, and Structural Characterization of Lysine Covalent BH3 Peptides Targeting Mcl-1

“…Our considerations in designing covalent peptides followed the general strategies we previously reported, where we targeted the ubiquitin ligase SIAH with a peptide derived by the protein Sip . Recently, we have studied the reactivity of aryl-fluorosulfates and aryl-sulfonyl fluorides ,,, as possible electrophiles to target surface Lys, Tyr, or His residues at protein–protein interfaces. In this present study, we examined the X-ray crystal structures of hMcl-1 in complex with various BH3 peptides, so as to design possible covalent agents.…”

“…Hence, to further improve the affinity for compound 12 for Mcl-1 via a possible covalent interaction with Lys 234, we first introduced a l -diamino-propionic acid ( l -Dap) in lieu of its N-terminal Ala residue, and its side chain amine was subsequently coupled with various fluoro-sulfonyl-benzoic acids (compounds 13 – 16 ; Table ). Incorporation of the sulfonyl fluorides was possible by using an ivDde protected l -Dap as we reported recently. ,,, The electrophiles in agents 13 - 16 were chosen based on our recent studies aimed at identifying “warheads” that presented the proper balance between buffer stability and reactivity. , Accordingly, aqueous stability studies at pH = 7.5 using one-dimensional 1 H NMR indicated that the half-life for compounds 15 or 16 was ≫3 h 30 min for each agent at room temperature (Figure S5). In particular, compound 15 remained >80% intact after 3 h 30 min in aqueous buffer, while compound 16 remained nearly completely intact under the same experimental conditions (Figure S5).…”

“…The success of the approach in targeting Cys is undoubtedly due mostly to the discovery that certain electrophiles based on acryl amides possess the proper balance between Cys reactivity and stability in buffer, plasma, and in vivo to be used as effective “warheads”. Recently, several studies, − including those from our laboratory, ,− have identified aryl-fluorosulfates or aryl-sulfonyl fluorides as chemical moieties that could function as possible electrophiles for targeting Lys, Tyr, or His residues. Stability and cell permeability studies suggested that these moieties could present a proper balance of stability in buffers and in cells to be used as pharmacological tools or could even in the future be introduced into therapeutic agents.…”

“…To this end, we previously developed a variety of novel ligand discovery strategies including HTS by NMR and related approaches, − while we more recently focused on strategies to derive covalent PPIs antagonists. − In this present study, we targeted Mcl-1, an anti-apoptotic Bcl-2 protein. − Our earlier studies in this field included the derivation of semisynthetic pan-Bcl-2 antagonists, which have been used to address the role of Mcl-1 in various cellular assays. − A major goal of our research is to derive potent and selective pharmacological tools that can be used to dissect the role of each of the Bcl-2 proteins in apoptosis and cancer resistance and that can be additionally used as stepping stones for the design of future therapeutic agents. While this goal may appear arduous on many levels, we recently developed innovative strategies to covalently target Lys residues to help overcome the challenge of deriving potent and selective antagonists of PPIs, − and we deployed this Lys-covalent binding strategy here to target hMcl-1.…”

DABSO as a SO2 gas surrogate in the synthesis of organic structures

“…[5][6][7][8][9][10][11] There is a surge of attention paid to innovative and improved synthetic techniques for introduction of sulfur including functional groups, like sulfones and sulfonamides, in the structure of agrochemicals, medicines, and other organic materials. [12][13][14][15][16][17][18] Thus, the development of convenient and stable SO 2 surrogates is thought to be a key driver in the revitalization of sulfur-based organic synthesis research. Hence, 1,4-diazabicyclo[2.2.2]octane bis(sulfur dioxide), abbreviated as DABSO, has been manufactured 19 to use it as a reagent for sulfonating organic structures.…”